CN1020471C

CN1020471C - Quality improvement of alcoholic liquors

Info

Publication number: CN1020471C
Application number: CN87106909A
Authority: CN
Inventors: 柿本茂八; 隅野靖弘; 山田秀明; 今安聪; 市川英治; 水津哲义
Original assignee: Gekkeikan Sake Co Ltd; Takeda Pharmaceutical Co Ltd
Current assignee: Gekkeikan Sake Co Ltd; Kirin Food-Tech Co Ltd
Priority date: 1986-10-14
Filing date: 1987-10-14
Publication date: 1993-05-05
Anticipated expiration: 2002-10-14
Also published as: EP0266088A1; AU7933987A; DE3786391T2; MX169236B; CA1311701C; CN87106909A; KR950006812B1; BR8705459A; KR880005252A; NZ222144A; EP0266088B1; DE3786391D1; US4844911A; AU598305B2; ES2056826T3

Abstract

Alcoholic liquors containing carbamide are treated with the urease which has the optimal pH for the activity in an acid region, especially in the region of pH 2 to 5. By such treatment, the carbamide can be completely decomposed and removed from the alcoholic liquors in a much smaller amount of urease and in a short time as compared with the conventional treatment according to a urease having the optimal pH in the neutral to alkaline region. This method is more advantageous from the viewpoint of practical use and the quality of the thus obtained alcoholic liquors are superior to that obtained by the conventional treatment.

Description

Quality improvement of alcoholic liquors

The present invention relates to improve the liquor method for quality.

All fermented products comprise that refining pure mellow wine, beer, grape wine and samshu and finished wort (in order to the raw material of distillation whisky, brandy, shochu etc.) etc. all contain urea, this is the major cause that makes liquor rotten, when liquor carried out pasteurization or standing storage, urea made liquor lose Wei Dao And to make their local flavor rotten.In order to remove urea, a kind of known method is to add urase preparation ， And to make it reaction (No. 20830/1981, Japanese patent gazette) under 10～20 ℃ low temperature in liquor or finished wort.

Urase (E, C, 3,5,1,5) is a kind of urea to be decomposed into the enzyme of ammonia and carbon dioxide gas, and it is distributed widely in occurring in nature, for example plant, animal and microorganism.Conventional urase from sword bean (Canavalia Adans) and Pasteur genus bacillus (Bacillus pasteuri) is carrying out suitability for industrialized production for practical application.

The best pH value of above-mentioned urase product in neutrality to basic region, when pH value during at acidic region, not only reaction is difficult to carry out, and enzyme is easy to passivation, when temperature of reaction contains organic solvent (as ethanol time) more than room temperature or in the reaction mixture that reacts, this point is particularly remarkable.Therefore, No. 20830/1981 disclosed method of Japanese patent gazette is defective, in order to remove the urea that contains in the refining pure mellow wine of about 20% alcoholic acid, this method must add the urases from sword bean or bacterium (best pH6-8) in a large number, and reacts to take long to and carry out under 10-20 ℃ low temperature; Therefore, it seems that this method is unsafty for the production of liquor with industrialized viewpoint.

The present inventor thinks, in order to produce high-quality liquor, prepares a kind of even also can Bao in containing the acid liquor of alcoholic acid keeps steady that to Ding the urase that And has a good action be very necessary.As their result of study, they find, handle at the urase from lactic-acid-bacterium of acidic region (pH2-5) with optimal ph, as long as add a small amount of urase, just can be at short notice with in addition under comparatively high temps, remove urea in the liquor by decomposition.After further studying, the present inventor has finished the present invention.

In other words, the present invention relates to the improvement of liquor quality, it is characterized in that handling liquor with acid urease.

The used acid urease of the present invention is meant a kind of like this enzyme, it produces 2 moles of ammonia and 1 mole of carbon dioxide gas from 1 mole of urea and 1 mole of water, its active optimal ph scope is at pH-5 pH2-4.5 preferably, general aspects about this enzyme is unqualified, for example the characteristic of PH stability, optimum temps, thermostability, Substratspezifitaet, inhibitor, Km value and molecular weight.

Acid urease is generated by the bacterial strain that produces acid urease usually.This bacterial strain is those so-called " lactic-acid-bacteriums " preferably, for example belong to the bacterial strain of dependent of dead military hero down: streptococcus, plane Coccus, leuconos toc, lactobacillus and genus bifidobacterium.Preferably use following representational bacterial classification: streptococcus faecalis (Streptococcus faecium), Streptococcus mitis (S.mitis), lactobacterium casei cheese mutation (Lactobacillus casei var casei) Steeptococcus mitior, streptococcus bovis (S, bovis), lactobacillus fermentum (L.fermentum) child's bifidus bacillus (Bifidobacterium infantis) Bifidobacterium choerinum (B.suis) and Bifidobacterium choerinum, but unqualified about bacterial strain; Even from milk-product, the fresh separated thing of the food of soil, corrupt acid change, the organ of animal and secretory product etc. also can use, as long as they can produce acid urease.Handle the artificial variation's strain that obtains from these bacterial strains by uv-radiation or mutagenic compound in addition, and other bacterial strains that the reorganization by artificial isolating gene fragment obtains also can use, and active expression is essential to these gene fragments for described acid urease.

Specifically, the bacterial strain that produces acid urease comprises: lactobacillus fermentum JCM5867(IFO 14511CCTCC M87078), lactobacillus fermentum JCM 5868(IFO 14512 CCTCC M87079), lactobacillus fermentum JCM 5869(IFO 14513, CCTCC M87080), Streptococcus mitior PG-154(IFO 14633, CCTCC M87083), streptococcus bovis PG-186(IFO 14634, CCTCC M87084) and Bifidobacterium choerinum PG-196(IFO 14635, CCTCC M87082).The IFO number is meant the preserving number in Osaka fermentation research institute (IFO), and the CCTCC number is meant at Chinese Wuhan typical case's training thing collects the preserving number at center.

The date that these microorganisms are pointed out in following table is preserved in CCTCC.

Microorganism is at the preservation date preserving number of CCTCC

Lactobacillus fermentum JCM5867 1987.10.4 CCTCC M87078

Lactobacillus fermentum JCM5868 1987.10.4 CCTCC M87079

Lactobacillus fermentum JCM5869 1987.10.4 CCTCC M87080

S.mitior PG-154 1987.10.4 CCTCC M87083

Streptococcus bovis PG-186 1987.10.4 CCTCC M87084

B.choerinum PG-196 1987.10.4 CCTCC M87082

Lactobacillus fermentum JCM5867, lactobacillus fermentum JCM5868 and lactobacillus fermentum JCM5869 list " the 13rd phase research communication (1985～1986 annual report) 1987 " in above-mentioned IFO number, and this book is published by IFO.

Above-mentioned six kinds of microorganisms have following bacteriology character.

Bacterial strain

Character JCM5867 JCM5868 JCM5869

Cell shape quarter butt quarter butt quarter butt

（0.6-1.2×1.0-2.0）（0.6-1.2×1.0-2.0）（0.6-1.2×

1.0-1.2

Motility---

Son formation---

Gramstaining+++

30～40 ℃ 30～40 ℃ 30～40 ℃ of best production temperature

45 ℃ growths+++

15 ℃ growths---

Oxygen demand facultative anaerobe facultative anaerobe facultative anaerobe

The fermentation of the fermentation of oxidation-fermentation test fermentation

Fermented type generates from glucose in a large number from a large amount of the generation from a large amount of generation of glucose of glucose

DL-lactic acid and ethanol DL-lactic acid and ethanol DL-lactic acid and ethanol

Gas forms

Glucose+++

Grape acid+++

Acid forms

Glucose+++

Glyconic acid+++

N.F,USP MANNITOL---

Saligenin---

Raffinose+++

Galactitol---

Inositol---

Lactose+++

Melizitose---

Melibiose+++

Maltose+++

Cellobiose---

Vitamin C2---

Glycerine---

Seminose---

Rhamnosyl---

Ribose+++

Pectinose---

Sorbyl alcohol---

Sucrose+++

Fructose+--

Semi-lactosi+++

Trehalose---

Wood sugar---

Catalase---

Oxydase---

Nitrate reduction---

Gelatine liquefication---

Auxotrophy VitB1 VitB1 VitB1

Calcium pantothenate calcium pantothenate calcium pantothenate

Nicotinic acid nicotinic acid nicotinic acid

Riboflavin

Bacterial strain

Property P G-196 PG-154 PG-186

Source pig duodenum pig duodenum hog middle

Cell shape quarter butt quarter butt sphere

（0.6-0.8× （0.8-1.0× （0.8-1.0×

1.0-1.5）μ 0.8-1.0）μ 0.8-1.0）μ

Motility---

Sporulation---

Gramstaining+++

Oxygen demand microaerobe facultative anaerobe facultative anaerobe

The L-lactic acid of the L-lactic acid homogeneous of the L-lactic acid homogeneous of fermented type homogeneous

Catalase---

Oxydase---

The nitrogen reduction---

Gelatine liquefication---

The starch hydrolysis+-+(faint)

MR test+(faint)-+

The VP test++ (faint)+(faint)

Indoles formation---

Hydrogen sulfide formation--ND ND

From arginine---

Form NH ₃

Litmus milk---

From glucose form gas---

The Vitamin C2 decomposition+--

Optimum growth temp (℃) 25-37 30-37 25-37

45 ℃ growths---

15 ℃ growths---

The growth of pH4.0---

The growth of pH9.6---

3% sodium-chlor water-soluble+--

Growth in the liquid

6.5% sodium-chlor---

Grow in the aqueous solution

The alpha hemolysis effect--+(faint)

The beta hemolysis effect---

Acid forms

Adonitol-ND ND

Pectinose+--

Arabitol-ND ND

Arbutin-+(faint)+

Cellobiose+++

Galactitol-ND ND

Fructose-++

Semi-lactosi+++

The grape hydrochlorate---

Glucose+++

Glycerine---

Inositol---

Inulin---

Lactose+++

Maltose+++

Seminose--+

N.F,USP MANNITOL---

Melizitose---

Melibiose+--

Alpha-Methyl Portugal+(faint) ND-ND

Glucosides

Raffinose++-

Rhamnosyl---

Ribose+(faint)--

Saligenin-++

Sorbyl alcohol---

Sorbose-ND ND

Starch+-+

Sucrose+(faint)++

Trehalose---

Wood sugar+--

Xylitol-ND ND

Dna content (%) 65.5 40.3 40.1

Peptidoglycan Lys ND ND

Type

Orn

Ser

Ala

Glu

In the superincumbent table, symbol " ND " is not meant and experimentizes that Lys, ASP, Ala, Orn, Ser and m-DAP represent Methionin, Aspartic Acid, L-Ala L-glutamic acid, ornithine, Serine and a diaminopimelic acid respectively.By above-mentioned bacteriology character and the description in " uncle Jie Shi classification bacteriology handbook, the 2nd volume " (1986) are compared, checked the taxonomic position of above-mentioned bacterial strains.As check result, all be positive though the PG-196 bacterial strain forms acid by pectinose, cellobiose, ribose and wood sugar etc., with it divide be still into microorganism Bifidobacterium choerinum suitable; Though PG-154 bacterial strain alpha hemolysis act as feminine gender, but still belong to Streptococcus mitior; Though the Vitamin C2 hydrolysis of PG-186 bacterial strain is negative, but still belongs to streptococcus bovis.

Acid urease static cultivation, shaking table cultivation, ventilation rotating and culturing or solid culture by routine from these bacterial strains carried out continuously or batch production.Used substratum is the conventional substratum that bacterium can grow therein.Carbon source preferably is selected from carbohydrate, fat, lipid acid and the alcohols that can assimilate, and they can use separately or be used in combination.Nitrogenous source comprises organic nitrogen source such as peptone, soyflour cottonseed meal, corn steep liquor, yeast extract paste, meat extract, malt extract and whey etc., and inorganic nitrogen-sourced for example ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate, after can using separately or combine valuably as required, they use.Except carbon source and nitrogenous source, preferably in substratum, add basic somatomedin or growth stimulant such as mineral substance, amino acid and VITAMIN.Sometimes add urea, thiocarbamide or its analogue to induce the formation of acid urease.In order to control pH value and the foam in the culturing process, it is effective adding suitable caustic solution, sodium carbonate solution or calcium salt soln or foam reducing composition.

Selected culture temperature is generally 15～50 ℃ preferably 25～40 ℃ in being suitable for the scope of used bacterial growth.Incubation time lasts till the production of enough bacterial growths and acid urease, normally 5～50 hours.

After cultivating under these conditions, contain acid urease in the bacterial cell usually.By centrifugal, precipitation, aggegation, from culture, collect viable cell by perforated film or ceramic filter etc., in the present invention, this cell can be used as the raw product of acid urease, and needn't be through any further processing or through lyophilize, spraying drying, acetone drying etc.It also is feasible making described enzyme dissolving, (use separately or be used in combination and get final product) such as promptly molten by freezing, grinding, sonic treatment osmotic shock, N,O-Diacetylmuramidase, tensio-active agents handles cell, the routine techniques that is used in combination various suitable enzyme purifications then carries out purifying, for example protamine is handled, is saltoutd, organic solvent processing, isoelectric precipitation, electrophoresis, ion exchange chromatography, gel-filtration, affinity chromatography and crystallization, than viable cell higher single-minded active enzyme raw product or purifying preparation are arranged thereby produce, they can be used in the method for the present invention.

The following describes the method for handling liquor with acid urease.

Be used to handle the enzyme form of liquor, it can be the bacterial cell that contains this enzyme, or the raw product or the purifying preparation of the acid urease that obtains of extraction and purification technique by routine, or by zymin being embedded in the cured article that obtains in natural polymer such as agar and carrageenan or synthetic polymer such as polyacrylamide and the urethane resin, or by being combined in a kind of carrier such as gac, pottery, dextran, agarose and related substance thereof and the porose cured article that obtains on glass.

The liquor that will handle with the inventive method is those beverages that contain urea, comprises that fermented product is as refining pure mellow wine, beer, grape wine, fruit wine, samshu, whisky wort, shouhu wort (Shouhu is a kind of Japanese ardent spirits) and brandy wort and intermediate product thereof etc.For example, available the inventive method is handled the unpurified pure mellow wine that is contained in behind Japanese sake, finished wort, the wort press filtration in the drum, give birth to the anticorrosion pure mellow wine after alcohol, the pasteurization, the refining pure mellow wine before the bottling etc., but, be preferably in the described enzyme of the preceding adding of pasteurization and handle.

When handling these enzyme liquors with acid urease, the acid urease that adds 0.00001 unit/ml to 1 unit/ml is very useful, especially the 0.0001 unit/ml of unit to 0.1.One unit be meant in the unit time (minute) decompose urea and discharge the required enzyme amount of 1 micromole's ammonia.The writing 1U of this paper back one unit.

The temperature of handling liquor is generally 0 ℃-80 ℃, preferably 10 ℃～60 ℃.PH is 2-7, better is 3-5.Treatment time will last till the urea that is enough to remove in the liquor, is generally 20 minutes-200 days, and more frequent is 5 hours to 120 days.

Also can handle liquor with acid urease, promptly in the ethanol fermentation process of carrying out, the micro-organisms living cell that produces acid urease be cultivated together for the production liquor by following method.

Any time before finishing ethanol fermentation, all can add the bacterium that produces acid urease.Preferably the bacterium of acid urease is produced in inoculation, and it is grown in the process of producing the yeast wort or in the process of preparation saccharification mixed solution, makes its experience Primary Fermentation (ethanol fermentation) then according to conventional methods.Can when Primary Fermentation suitable, plant And indirectly and cultivate the bacterium that produces acid urease, preferably before the mid-term of whole yeast phase.

When the bacterium that produces acid urease is grown, should before adding yeast usually, inoculate the bacterium that produces acid urease in the yeast wort of liquor, after the bacterium that produces acid urease fully grows, inoculate yeast; Prepare the saccharifying that seed Mai Ya Zhi And is used for starch then according to conventional methods.If the bacterium that produces acid urease is grown in the coarse raw materials of coarse raw materials or saccharification, the bacterium ， And that then tackles the coarse raw materials inoculation product acid urease of coarse raw materials (as steaming rice, bent rice (a kind of culture that steams the Aspergirus oryzae on the rice), Fructus Hordei Germinatus, barley juice, Sucus Vitis viniferae, starch) or saccharification fully is being used for mashing after the growth.

Produce the cultivation Wen Du And indefinite of acid urease bacterium, but preferably 28-40 ℃.Produce the growing number indefinite of acid urease bacterium, but preferably 10 ⁸/ ml or more.The coarse raw materials that contains the seed wort that produces the acid urease bacterium or coarse raw materials or saccharification can be used for finishing any saccharification stage before the ethanol fermentation or any time of saccharifying.For example produce when making with extra care pure mellow wine, they can be at the initial stage of saccharifying, and mid-term or termination add fashionable use, or any time in the saccharifying uses.The adding Liang And indefinite that contains the coarse raw materials of the seed wort that produces the acid urease bacterium or coarse raw materials or saccharification, but 3-15% preferably.

When in the seed wort, adding the bacterium that produces acid urease, if this bacterium is the acid-producing bacteria strain then there is no need to add acid, yet, if this bacterium does not produce acid, then will add acid (for example lactic acid) before adding yeast, add-on almost is equal to the amount that is generally used for the seed wort.

Ethanol fermentation of the present invention can carry out (temperature, time etc.) under normal condition, in process subsequently, for example filter, compress filtration, pasteurization, storage, ageing and distillation etc., does not require used condition is changed.

With use the ordinary method of handling to alkaline urase from the neutrality of sword bean and compare, the method that the present invention handles liquor with acid urease is more superior economically, because required urase quantity wants much less that is to say, method of the present invention only needs unit of enzyme or the enzyme of about 1/10-1/100 weight or the amount still less of about 1/100-1/10000 of ordinary method, just can remove urea fully by decomposing.Therefore, be actually negligible owing to remain in the corruption that the urase in the liquor causes after handling.This compares with ordinary method is a more useful aspect.

In addition, utilize method of the present invention, the decomposition of urea under higher temperature in a short time and even at acid pH 3-5(this is common for liquor) condition under carry out; Urea can be decomposed by high-level efficiency, and this is very favourable to suitability for industrialized production.

The present invention gives more specific description with example below.These examples provide them as just example and never limit the scope of the invention.

The activity of acid urease is carried out colorimetric estimation by general salt method in the culture, make the culture cell of centrifugal collection place aseptic deionized water, getting the cell suspending liquid that a volume suitably dilutes mixes with isopyknic 0.2M citrate buffer (pH4.0) that contains urea, make mixture at 37 ℃ of reactions 30 minutes, the ammonia that colorimetric estimation then produced.Active in unit representation, a unit (1U) be meant the unit time (minute) produce the enzyme amount of micromole's ammonia.

Example 1

To lactobacillus fermentum JCM5867(IFO 14511, CCTCC M87078) carries out stab culture, substratum consists of: 0.5% meat extract, 0.5% salt, 1.0% lime carbonate and 1.5% agar, after the cultivation it is inoculated in the Erlenmeeyer culturing bottle of 200ml, the aseptic culture medium (with the neutralization of 30% caustic soda) that contains 50ml pH7.0 in each bottle, consisting of of this substratum: 2.0% glucose, 2.0% sodium acetate, anhydrous, 1.0% albumen freezes, 1.0 meat extract, 0.2% yeast extract paste, 0.5% salt and 0.005% sal epsom (every mole contains about 4 moles of crystal water), under 37 ℃, leave standstill then and cultivated 24 hours.The inoculum that produces in each bottle is transferred among of two 2 liters of Erlenmeyer culturing bottles, contains 1 liter of aseptic culture medium identical with above-mentioned composition in each bottle, leaves standstill cultivation 24 hours then under 37 ℃.Urease activity in these nutrient solutions is 0.1U/ml.

By the cell in the centrifugal collection nutrient solution, (pH7.2) washes twice with the 0.05M phosphate buffered saline buffer, is suspended in the solution that contains 30ml0.05M phosphoric acid buffer (pH7.2) 0.02M EDTA and 0.01M dithiothreitol (DTT), carries out sonic treatment; With ammonium sulfate supernatant liquor is separated, be collected in the precipitation that obtains under the 40%-70% saturation ratio, it is dissolved in the 10ml0.05M phosphoric acid buffer, the liquid of dialysing, frost drying, the result obtains the thick enzyme powder of 260.4mg acid urease.Urease activity is 0.5U/mg, and purification efficiency is 65.1%.

The general aspects of thick enzyme powder is as follows:

Best pH pH2-4.5

Optimum temps 60-70 ℃

PH stability is when pH2-10, and it is stable to handle maintenance in 2 hours down at 37 ℃

Thermostability is when pH4, and maintenance in 2 hours is stable handling below 60 ℃

Then, thick enzyme powder being dissolved in the refining pure mellow wine (contains 20% ethanol and 30ppm urea, pH4.3) makes its concentration be approximately 0.02U/ml or 0.1U/ml, remain on the urea that makes under 30 ℃ in the refining pure mellow wine and decompose; The results are shown in the table 1.With the commercially available jack bean urease preparation that gets in contrast with 10U/ml.Urase with sword bean was handled 5 days with 10U/ml, and the urea in the refining pure mellow wine has approximately only reduced half, and only handles one day with the acid urease that is low to moderate 0.1U/ml, and urea has been decomposed fully.

By the activity of Nitroprusside ion colorimetric method for determining acid urease and jack bean urease, be determined at the ammonia that pH4.0 and pH7.0 produce respectively.By the urea content in the refining pure mellow wine of enzymatic assays, use to rely on NADP ⁺Glutamate dehydrogenase measure with urase and decompose the ammonia that urea produces.

These measuring methods also are applicable to following example.

Table 1

The processing fate of used urase

Kind and quantity 0 day 1 day 2 days 5 days

ppm ppm ppm ppm

0.02U/ml acid urease 30 530

0.1U/ml acid urease 30 000

10.0U/ml jack bean urease 30 18 14 13

Example 2

In living pure mellow wine, (contain 20% ethanol and 30ppm urea pH4.3) and add the thick enzyme powder of the acid urease that obtains in the example 1, make the activity of acid urease be approximately 0.01U/ml or 0.03U/ml, remain on 10 ℃ or 15 ℃ and decompose ureas down.As shown in table 2, after 10 ℃ are handled 8 days down, the urea in the living pure mellow wine is decomposed with the 0.003U/ml acid urease fully.Taste test shows that refining pure mellow wine (not handling with the thick enzyme powder of the acid urease) quality of the refining pure mellow wine comparison photograph after the processing is more superior, and ageusia still less.

Table 2

Temperature urase amount is handled fate

0 2 4 6 8

mU/ml ppm ppm ppm ppm ppm

10℃ 10 30 12 4 0 0

3 30 18 10 4 0

15℃ 10 30 8 0 0 0

3 30 14 8 3 3

Example 3

Same quadrat method cultivation and fermentation Bacterium lacticum JCM5867(IFO 14511 CCTCC M87078 as example 1), collecting cell from 4 liters of nutrient solutions that obtain produces the 2.0g stem cell behind the frost drying.The acid urease activity of these stem cells is 0.35U/ml.

Living pure mellow wine (contains 20% ethanol and 30ppm urea, pH4.3) 75 ℃ of following pasteurizations 1 minute, is cooled to 30 ℃ rapidly, to wherein adding above-mentioned stem cell, make the activity of acid urease be approximately 0.01U/ml or 0.003U/ml, remain under 30 ℃.Along with changing, the urea concentration of time is shown in Table 3.

Table 3

The urase amount is handled fate

0 4 6 10

10mU/ml ppm ppm ppm ppm

30 9 0 0

3 30 16 7 0

Example 4

Living pure mellow wine (contains 20% ethanol and 30ppm urea, pH4.3) 62 ℃ of following pasteurizations 15 minutes, be cooled to 55 ℃ rapidly, the thick enzyme powder that adds the acid urease that obtains in the example 1 makes it aseptic dissolving, make its activity be approximately 0.003U/ml, be cooled to room temperature then rapidly, under the normal condition of refining pure mellow wine, preserve.Along with changing, the urea concentration of time is shown in Table 4.

Table 4

The urase amount is handled fate

0 4 7 10

3mU/ml 30ppm 12ppm 4ppm 0ppm

Mixture temperature (℃) 20 21 23 24

Example 5

After fermentation is finished, in the finished wort of pure mellow wine, (contain 18% ethanol and 30ppm urea, pH4.2) add the thick enzyme powder of the acid urease that example 1 obtains, make its activity be approximately 0.01U/ml, remain on 13 ℃, be filtered in the drum later in three days.Urea in the finished wort as shown in table 5 decomposed in 3 days fully.After finished wort filters, in the pure mellow wine of drum, also detect less than urea.

Table 5

Handle fate

Pure mellow wine in 0123 drums

Urea (ppm) 30 18 800

Example 6

In beer, (contain 4.2% ethanol and 5.1ppm urea, pH4.2) dissolve in the thick enzyme powder of the acid urease that example 1 obtains, make its concentration be approximately 0.003U/ml, kept 3 days down at 10 ℃; Urea concentration variation in the beer is shown in Table 6.Urea can decompose in 3 days fully.Taste test shows that the beer of handling is not than better with the beer of acid urease processing like this, and ageusia still less.

Table 6

Handle fate

0 1 2 3

Urea (ppm) 5.1 3.0 1.5 0

Example 7

With described with quadrat method cultivation and fermentation Bacterium lacticum JCM5867(IFO14511, CCTCC M87078 as example 1), make 4 liters of medium centrifugals that obtain and collecting cell produces the 2.0g stem cell behind the frost drying.The activity of acid urease is 0.35U/ml in these stem cells.These stem cells are to float in the refining pure mellow wine (to contain 20% ethanol and 30ppm urea, pH4.3), kept 6 hours down at 50 ℃, make the urea completely dissolve with the concentration of 0.5mg/ml.

Example 8

In grape wine, (contain 12.1% ethanol and 6.2ppm urea, pH3.7) add the stem cell that contains acid urease that example 7 obtains, make its activity be approximately 0.03U/ml and kept 5 days down, make the urea completely dissolve in the grape wine at 15 ℃.

From grape wine, remove cell then, compare by taste test and the grape wine of not handling with cell; Better with the grape wine that cell is handled, ageusia still less.

Example 9

(contain 5.3% ethanol and 5.0ppm urea, pH4.3) and add the thick enzyme powder of the acid urease that example 1 obtains in the whisky wort, concentration is 0.01U/ml, keeps 24 hours down at 22 ℃, and the urea in the whisky wort is decomposed fully.

Whisky wort of Chu Liing and untreated whisky wort like this, every kind is all distilled twice in the glass distiller, to obtain containing 50% alcoholic acid whisky product.Compare these two kinds of products with taste test; Whisky product after the processing is better, and ageusia still less.

Example 10

(containing 17.5%) ethanol and 30ppm urea in rice wine wort (rice shochu mash), pH3.8) add the stem cell that contains acid urease that example 7 obtains, make its activity be approximately 0.03U/ml, kept 2 days down, make the urea completely dissolve in the rice wine wort at 15 ℃.

The rice wine wort that adds the rice wine wort of cell and do not add cell under reduced pressure distills, and contains 40% alcoholic acid rice wine product with generation.Compare these two kinds of products by taste test; Product after the processing is better, and ageusia still less.

Example 11

To lactobacillus fermentum JCM5867(IFO 14511, CCTCC M87078) carries out stab culture, substratum consists of: 0.5% glucose, 1.0% albumen freezes, 1.0% yeast extract paste, 0.5% meat extract, 0.5% salt, 1.0% lime carbonate and 1.5% agar, inoculation is gone in two 1 liter the Erlenmeyer culturing bottle then, the aseptic culture medium (with the neutralization of 30% caustic soda) that contains 500ml pH7.0 in each bottle, substratum consists of: 2.0% glucose, 2.0% sodium acetate, anhydrous, 1.0% albumen freezes, 1.0% meat extract, 0.2% yeast extract paste, 0.5% salt and 0.005% sal epsom (every mole contains about 4 moles of crystal water), under 37 ℃, leave standstill then and cultivated 24 hours, make viable count become 2.4 * 10g/ml.The inoculum that produces centrifugal 10 minutes with 10000rpm, the cell of collection washs the back recentrifuge with the 500ml sterilized water.Cell suspension after the washing is in the 10ml sterilized water.

In steaming rice and distillers yeast (be equivalent to 30kg and grind good rice and the bent rice of 30kg) mixture, add 120 premium on currency, keep making it in 5 hours saccharification at 55 ℃.Make the saccharification mixed solution be heated to 70 ℃ and keep 5 minutes, be cooled to 35 ℃ then to carry out disinfection.The suspension liquid of living cell that adds the above-mentioned lactobacillus fermentum JCM5867 of 10ml therein.Make mixture be incubated 2 days down then at 35 ℃.The lactic-acid-bacterium number is controlled at 2 * 10g/ml.Mixture is cooled to 28 ℃ and inoculates saccharomyces sake Kyokai-7 therein, makes its concentration be approximately 2 * 10 ⁷/ ml is incubated after 4 days wort is given to add for the first time, makes its sedimentation one day and does not add (odori) and give second and adding for the third time then; Make then under its positive conventional temperature variation condition and ferment.Then, gave the 4th time to wort and add in the time of the 19th day, ethanol and unpurified pure mellow wine are filled in the drum.Table 7 has been listed the quantity of each each component of saccharification stage in this example.Table 8 has been summarized the variation in the seed wort.Table 9 has provided the analytical results that is filled into the pure mellow wine in the drum.From table, can be clear that, handle in the finished wort for preparing after the seed wort with the lactic-acid-bacterium that produces acid urease and almost detect, because it has been decomposed by acid urease and can detect urea in contrast less than urea.Quality product is also better, and more horn of plenty is shone in the taste comparison.

The amount (kg) of table 7, saccharifying each component in each stage

Saccharomyces sake the 4th time for the third time for the first time for the second time

Culture adds total

Total rice 60 130 255 475 80 1000

Steam rice 30 95 200 395 80 800

Bent rice 30 35 55 80 200

Water 120 100 320 620 150 1310

Table 8, seed wort are along with the variation of time

Fate 12345 behind the adding yeast

Baume(Be) contrast 11.3 10.1 8.9 7.7 6.1

The present invention 13.5 10.5 7.8 6.6 6.0

Ethanol (Alc) (%) contrasts 1.5 3.6 5.8 7.0

The present invention 1.5 5.0 6.3 6.7

Acid (T, A) contrast 4.10 5.10 5.75 6.32 7.10

The present invention 5.58 8.28 10.18 9.85 9.90

Amino acid acidity (A.A.) contrast 2.0 1.71 1.85 1.80 1.90

The present invention 5.30 4.57 3.77 3.60 3.45

Lactobacillus fermentum contrasts 0.0 0.0 0.0 0.0 0.0

JCM5867 counting (* 10 ⁶) the present invention 380 400 400 400 400

Lactic acid contrasts 510 615 700 800 920

(ppm) the present invention 1,094 1,157 1,230 1,270 1260

Table 9, be filtered to the analytical results of the pure mellow wine in the drum

Be Alc T.A A.A urea (ppm)

Contrast-2.3 19.9 1.80 1.75 15.3

The present invention-2.0 19.9 1.85 1.82 0.0

Example 12

In the mixture that steams rice and distillers yeast, (be equivalent to 60kg and grind good rice and the bent rice of 40kg) add 180 premium on currency, keep 5 hours to carry out saccharification at 55 ℃.The saccharification mixture keeps carrying out pasteurization in 5 minutes by being heated to 70 ℃ And, is cooled to 35 ℃ then.The suspension liquid of living cell of the lactobacillus fermentum JCM 5867 that adding 40ml example 11 obtains in this refrigerative saccharification mixture.Make mixture be incubated 2 days down then at 35 ℃.The counting of lactic-acid-bacterium is controlled at 2 * 10 ⁸/ ml.The total amount of this saccharification mixture was added to (the 4th adding) in the wort at the 12nd day, gave the 5th adding at the 18th day and add ethanol, and pure mellow wine is filled in the drum.The schedule of quantities 11 that table 10 has been listed each each component of saccharification stage in this example has provided in the wort urea content over time.The table in back shows, after the 4th of handling with the lactic-acid-bacterium that produces acid urease was added wort, urea content wherein promptly began decline after adding, become 0 in the time of in being filled into drum, and this shows that urea is decomposed by acid urease.Pure mellow wine in drum also detects less than urea.Taste test shows that the pure mellow wine quality in the drum is splendid, has dense pure and fresh taste.

The quantity (kg) of table 10, each each component of stage of saccharifying

Saccharomyces sake is the 4th order five times for the third time for the first time for the second time

The culture adding amounts to

Total rice 50 125 235 430 100 60 1000

Bent rice 25 35 45 60 40 205

Steam rice 25 90 190 370 60 60 795

Water 100 100 290 550 180 120 1340

The variation of urea content (ppm) in table 11, the wort

The wort age (my god) pure mellow wine in 12 13 14 16 18 drums

Contrast 21 24 26 28 30 24

The present invention 21 11 3 1.2 00

Example 13

(Nissui Seiyaku Co.Japan) cultivates Streptococcus mitior PG-154(IFO 14633 CCTCC M87083 on the semi-solid commercial product culture medium of GAM), it is inoculated in the Erlenmeyer culturing bottle of 200ml, (with the neutralization of 30% caustic alkali) the aseptic seed culture medium that wherein contains 50ml pH7.0, substratum consists of: 3% glucose, 1.5% albumen freezes, 1% meat extract, 0.8% yeast extract paste, 0.5% salt, 0.2% sodium acetate, anhydrous, 0.005% sal epsom (every mole approximately contains 4 moles of crystal water) and 0.001% rose vitriol (7 molecular water compound) leave standstill it and cultivated 24 hours under 34 ℃.Get in the Erlenmeyer culturing bottle that this inoculum of 5ml is transferred to 200ml, wherein contain the same aseptic culture medium of forming of 100ml, under 32 ℃, leave standstill and cultivated 2 days.Measure the activity of acid urease in this culture, measuring activity is 0.6U/ml.

Get the above-mentioned culture that obtains of 5ml and wash cell then with water with collecting cell with centrifugal 10 minutes of 3000rpm, centrifugal, be suspended in the 1ml sterilized water, adding 50ml Yi Ding Chun And kept 15 minutes down at 50 ℃, thereby produce a kind of enzyme solution, utilize the 0.2M citrate buffer to measure its best pH.The results are shown in the table 12.It is as shown in the table, and all bacterial strains of the present invention have intensive urea degrading activity in acid range.

Table 12

pH 3 4 5 6 7 8 9

Relative reactivity (%) 68 100 98 67 26 14 13

Centrifugal collecting cell from the 80ml culture is dipped in 50% ethanol and sterilizes, Li Xin And frost drying.The heavy 16.9mg of the stem cell that obtains, the acid urease activity is 1.44U/mg.

Stem cell is added in the refining pure mellow wine (contains 20% ethanol and 30ppm urea, pH4.3) make the acid urease activity be approximately 0.01U/ml, remain on 30 ℃ of ureas that decompose down in the refining pure mellow wine; The results are shown in the table 13.

Table 13

Handle fate 0246 10

Urea (ppm) 30 11 200

Example 14

The listed bacterial strain of table 14 below (Nissui Seiyaku Co.Japan) cultivates in the semi-solid commercial product culture medium of GAM, be inoculated in the Erlenmeyer culturing bottle of 200ml, contain the aseptic GAM commercial product culture medium of 50ml (Nissui Seiyaku Co.Japan) in each bottle, it is left standstill under 34 ℃ cultivated 24 hours.From every kind of inoculum that obtains like this, get in the Erlenmeyer culturing bottle that 5ml is transferred to 200ml, contain the 100ml aseptic culture medium in each bottle, this substratum is to obtain by add 0.005% sal epsom (every mole contains about 4 moles of crystal water) in same GAM commercial product culture medium, it is left standstill under 32 ℃ cultivated 3 days.The acid urease activity of measuring these cultures the results are shown in the table 14.

Table 14

Acid in the strain culture

Urease activity (U/ml)

Streptococcus bovis PG-186(IFO 14634 CCTCC M87084) 0.4

Bifidobacterium choerinum PG-196 1.2

（IFO 14635、CCTCC M87082）

Get the culture that 5ml obtains by aforesaid method, with collecting cell, wash with water with centrifugal 10 minutes of 3000rpm, centrifugal, be suspended in the 1ml sterilized water, add 50ml isopropylcarbinol ， And therein and kept 15 minutes down at 50 ℃; Utilize the 0.2M citrate buffer to measure the optimal ph of gained enzyme solution.The results are shown in the table 15.It is as shown in the table, and these bacterial strains of the present invention have intensive urea degrading activity in acid range.

Respectively get 80ml by PG-186 and PG-196 strain culture that aforesaid method obtains,, be dipped in 50% ethanol 4 hours with sterilization, Li Xin And lyophilize by centrifugal collecting cell.Heavy respectively 30.2mg of the stem cell that obtains like this and 64.0mg, the acid urease activity is respectively 0.38U/mg and 0.6U/mg.

Stem cell is added in the refining pure mellow wine (contains 20% ethanol and 30ppm urea, pH4.3) make its acid urease activity be approximately 0.01U/ml, remain under 30 ℃ to decompose the urea in the refining pure mellow wine.The results are shown in the table 16.

Table 15

Relative reactivity (%)

PH PG-186 PG-196

3 40 35

3 85 100

5 100 30

6 70 35

7 35 23

8 17 38

9 15 35

Table 16

Handle fate

Urase originates 0246 10

PG-186 30 10 0 0 0

PG-196 30 18 8 1 0

Claims

1, a kind of improvement liquor method for quality, this method comprises by the acid urease that adds 0.00001 unit/ml to 1 unit/ml in liquor handles the liquor that contains urea, described acid urease is the enzyme that produces 2 moles of ammonia and 1 mole of carbon dioxide gas from 1 mole of urea and 1 mole of water, and its active best PH is in acid range.

2, according to the liquor that the process of claim 1 wherein be produce by fermenting process and comprise product in the middle of it.

3, according to the method for claim 2, liquor wherein is pure mellow wine, beer, grape wine, fruit wine, samshu, whisky wort, sake wine (shochu) wort, brandy wort.

4, be pH2-5 according to the acid range that the process of claim 1 wherein.

5, handle with the acid urease of the bacterial cell form that contains described urase or the raw product or the purifying preparation of acid urease according to the liquor that the process of claim 1 wherein.

6, according to processing such as following the carrying out that the process of claim 1 wherein: make the viable cell that produces the acid urease microorganism in the ethanol fermentation process of liquor production, carry out common cultivation.

7, method according to claim 6, the microorganism of product acid urease wherein is lactobacillus fermentum JCM5867(IFO14511, FERM BP-1444, CCTCCM87078), lactobacillus fermentum JCM5868(IFO14512, FERM BP-1445, CCTCCM87079), lactobacillus fermentum JCM5869(IFO14513, FERM BP-1446, CCTCC M87080), Streptococcus mitior PG-154(IFO14633, FERM BP-1448, CCTCC M87083), streptococcus bovis PG-186(IFO14634, FERMBP-1449, CCTCC M87084) or Bifidobacterium choerinum PG-196(IFO14635, FERM BP-1450, CCTCCM87082), they can produce acid urease.

8, according to the method for claim 6, the microorganism of product acid urease wherein inoculates in producing yeast wort process or in the process of preparation saccharification mixed solution and makes its growth, makes its experience main fermentation stage then.